Difference between revisions of "Part:BBa K3332044"
 
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<partinfo>BBa_K3332044 short</partinfo>
 
<partinfo>BBa_K3332044 short</partinfo>
  
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An improvement of BBa_K1334002 by both enhancing the strength and adding the binding sites. It's more sensitive to formaldehyde.We use it to test whether it has any improvement compared with BBa_K1334002
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An improvement of <partinfo>BBa_K1334002</partinfo> by replacing weak promoter with strong promoter <partinfo>BBa_J23100</partinfo> and adding the binding sites. It's more sensitive to formaldehyde than <partinfo>BBa_K1334002</partinfo>.
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===Usage and Biology===
 
===Usage and Biology===
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As a DNA binding protein, hxlR is the transcriptional activator of the hxlAB operon from ''Bacillus subtilis''. The possible mechanism of formaldehyde induced expression is that the formaldehyde changes the conformation of hxlR which stimulating RNA polymerase to open the transcription.
 
  
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There are two binding sites (BRH1, BRH2) of hxlR, which are necessary for formaldehyde induced expression. Through'' in vitro'' experiments, the higher the concentration of hxlR, the stronger the reaction intensity of the binding. Therefore, we enhanced the expression of hxlR to improve the sensitivity of formaldehyde promoter. J23100 is used in the formaldehyde_derivative-3 promoter to express the hxlR, as opposed to weak promoter on the registry formaldehyde promoter (BBa_ K1334002). In addition, there are two BRH1 and two BRH2 in the formaldehyde_derivative-3 promoter, which is only one in the registry formaldehyde promoter (BBa_ K1334002).
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As a DNA binding protein, hxlR serves as a transcriptional activator of the hxlAB operon from ''Bacillus subtilis''. The formaldehyde changes the conformation of hxlR, stimulating RNA polymerase to open the transcription.
  
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[[File:2042.fig.1.png|none|500px|caption]]
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It is reported that the reaction intensity of complex binding becomes stronger and stronger with the increasing of the concentration of hxlR. Similar to formaldehyde_derivative-1 promoter, the strong promoter <partinfo>BBa_J23100</partinfo> is used in the formaldehyde_derivative-3 promoter rather than the weak promoter to express hxlR. Meanwhile, there are two BRH1 and two BRH2 in the formaldehyde_derivative-3 promoter, both of which only has one copy in the registry promoter (<partinfo>BBa_K1334002</partinfo>).Thus, the improvement of formaldehyde_derivative-3 promoter is the combination of formaldehyde_derivative-1 promoter and formaldehyde_derivative-2 promoter.
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Fig.1 Circuit
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<html>
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    <figure>
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        <img src="https://2020.igem.org/wiki/images/0/0d/T--XMU-China--XMU-China_2020-pHCHO_improve_1%2C2%2C3.png" width="60%" style="float:center">
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        <figcaption>
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        <p style="font-size:1rem">
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        </p>
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        </figcaption>
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    </figure>
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</html>
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'''Fig 1.''' Different improvements of formaldehyde promoter
  
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To construct this part, we moved formaldehyde_derivative-3 promoter (BBa_ K3332044) and RBS_ECFP_T(BBa_E0420) into the expression vector pSB1C3 by standard assembly. Then the ligation mixture was transformed into E. coli BL21（DE3） to compare the sensitivity of formaldehyde with other promoter.( formaldehyde_derivative-1 promoters, formaldehyde_derivative-2, formaldehyde promoter)
 
 
===Characterization===
 
===Characterization===
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We use Formaldehyde_derivative-3 promoter_B0034_E0020_B0015_pSB1C3 to characterize Formaldehyde_derivative-3 promoter.
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We use ECFP as the reporter gene to characterize the improvement.
  
 
The agarose gel electrophoresis images are below：
 
The agarose gel electrophoresis images are below：
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[[File:2044.fig.2.png|none|500px|caption]]
 
[[File:2044.fig.2.png|none|500px|caption]]
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Fig.2 Formaldehyde_derivative-3 promoter_B0034_E0020_B0015_pSB1C3(BBa_K3332092) digested by ''Xba'' I and ''Pst'' I (about 1543 bp)
 
  
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Protocol:
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'''Fig 2. '''Formaldehyde_derivative-3 promoter _B0034_E0020_B0015_pSB1C3(<partinfo>BBa_K3332092</partinfo>) digested by ''Xba'' I and ''Pst'' I (about 1543 bp)
  
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1.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12h.
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'''Protocol:'''
  
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2.Add 4mL of the above bacterial solution into 200 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.
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Part one: to compare the strength of weak promoter on the registry formaldehyde promoter (<partinfo>BBa_K1334002</partinfo>) and <partinfo>BBa_J23100</partinfo>
  
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3.Add 0.8mM formaldehyde into each group when OD600 increased to 0.6 and the culture condition is the same as before.
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1. Culture glycerol bacteria containing the corresponding plasmid in test tube for 12h.
  
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4.Then, sampling culture in 96-well plate reader every 3 hours to measure the fluorescence intensity (ECFP) and corresponding OD600, then calculate the fluorescence / OD value of each group.
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2. Add 4mL of the above bacterial solution into 200 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.
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3. Measure the fluorescence intensity (GFP) and corresponding OD<sub>600</sub>, then calculate the fluorescence / OD value of each group.
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Part two: to compare the sensitivity to formaldehyde of formaldehyde_derivative-3 promoter and formaldehyde promoter (<partinfo>BBa_K1334002</partinfo>)
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1. Culture glycerol bacteria containing the corresponding plasmid in test tube for 12 h.
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2. Add 4 ml of the above bacterial solution into 200 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.
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 +
3. Add 0.8 mM formaldehyde into each group when OD<sub>600</sub> increased to 0.6 and the culture condition is the same as before.
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4. Then, sampling culture in 96-well plate reader every 3 hours to measure the fluorescence intensity (ECFP) and corresponding OD<sub>600</sub>, then calculate the fluorescence / OD value of each group.
  
 
Here is the result:
 
Here is the result:
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[[File:T--XMU-BBa K3332092 .png|none|300px|caption]]
  
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[[File:2042.fig.4.png|none|500px|caption]]
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''' Fig 3.'''  Fluorescence intensity (GFP) /OD expressed by weak promoter, J23100 and blank. Data are collected and analyzed according to iGEM standard data analysis form after 6 hours of induction.
  
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Fig.3 The curve of fluorescence intensity (eCFP) /OD by pHCHO (BBa_ K1334002) and formaldehyde_derivative-3 promoter
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''' Note: '''  weak promoter is the promoter on the registry formaldehyde promoter (<partinfo>BBa_K1334002</partinfo>)
  
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We discovered that formaldehyde_derivative-3 promoter is more sensitive to the registry formaldehyde promoter (BBa_K1334002). What’s more, formaldehyde_derivative-3 promoter is the most sensitive promoter for formaldehyde. The detail about formaldehyde_derivative-1 promoter and formaldehyde_derivative-2 promoter can be seen in BBa_K3332042 and BBa_K3332043.
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[[File:2042.fig.4.png|none|400px|caption]]
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''' Fig 4.'''  The curve of fluorescence intensity (eCFP) /OD by pHCHO (BBa_K1334002) and formaldehyde_derivative-3 promoter.
  
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===Reference===
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In the former figure, we can see that the strong promoter <partinfo>BBa_J23100</partinfo> group has a higher relative fluorescence intensity than the weak promoter present in the registry formaldehyde promoter, and in the latter figure, we can compare formaldehyde_derivative-3 promoter with the other two derivative promoters. We can conclude from the latter figure that formaldehyde_derivative-3 promoter is more sensitive to formaldehyde than the registry one. What’s more, formaldehyde_derivative-3 promoter is the most sensitive promoter among them. The detail about formaldehyde_derivative-1 promoter and formaldehyde_derivative-2 promoter can be seen in <partinfo>BBa_K3332042</partinfo> and <partinfo>BBa_K3332043</partinfo>.
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[1]Yurimoto, H., Hirai, R., Matsuno, N., Yasueda, H., Kato, N. and Sakai, Y. (2005), HxlR, a member of the DUF24 protein family, is a DNA‐binding protein that acts as a positive regulator of the formaldehyde‐inducible hxlAB operon in Bacillus subtilis. Molecular Microbiology, 57: 511-519. doi:10.1111/j.1365-2958.2005.04702.x
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===Sequence and Features===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3332044 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3332044 SequenceAndFeatures</partinfo>
  
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<partinfo>BBa_K3332044 parameters</partinfo>
 
<partinfo>BBa_K3332044 parameters</partinfo>
 
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===Reference===
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[1]Yurimoto, H., Hirai, R., Matsuno, N., Yasueda, H., Kato, N. and Sakai, Y. (2005), HxlR, a member of the DUF24 protein family, is a DNA‐binding protein that acts as a positive regulator of the formaldehyde‐inducible hxlAB operon in Bacillus subtilis. Molecular Microbiology, 57: 511-519. doi:10.1111/j.1365-2958.2005.04702.x

Latest revision as of 21:35, 27 October 2020


Formaldehyde_derivative-3 promoter

An improvement of BBa_K1334002 by replacing weak promoter with strong promoter BBa_J23100 and adding the binding sites. It's more sensitive to formaldehyde than BBa_K1334002.

Usage and Biology

As a DNA binding protein, hxlR serves as a transcriptional activator of the hxlAB operon from Bacillus subtilis. The formaldehyde changes the conformation of hxlR, stimulating RNA polymerase to open the transcription.

It is reported that the reaction intensity of complex binding becomes stronger and stronger with the increasing of the concentration of hxlR. Similar to formaldehyde_derivative-1 promoter, the strong promoter BBa_J23100 is used in the formaldehyde_derivative-3 promoter rather than the weak promoter to express hxlR. Meanwhile, there are two BRH1 and two BRH2 in the formaldehyde_derivative-3 promoter, both of which only has one copy in the registry promoter (BBa_K1334002).Thus, the improvement of formaldehyde_derivative-3 promoter is the combination of formaldehyde_derivative-1 promoter and formaldehyde_derivative-2 promoter.

Fig 1. Different improvements of formaldehyde promoter

Characterization

We use ECFP as the reporter gene to characterize the improvement.

The agarose gel electrophoresis images are below：

caption

Fig 2. Formaldehyde_derivative-3 promoter _B0034_E0020_B0015_pSB1C3(BBa_K3332092) digested by Xba I and Pst I (about 1543 bp)

Protocol:

Part one: to compare the strength of weak promoter on the registry formaldehyde promoter (BBa_K1334002) and BBa_J23100

1. Culture glycerol bacteria containing the corresponding plasmid in test tube for 12h.

2. Add 4mL of the above bacterial solution into 200 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.

3. Measure the fluorescence intensity (GFP) and corresponding OD600, then calculate the fluorescence / OD value of each group.

Part two: to compare the sensitivity to formaldehyde of formaldehyde_derivative-3 promoter and formaldehyde promoter (BBa_K1334002)

1. Culture glycerol bacteria containing the corresponding plasmid in test tube for 12 h.

2. Add 4 ml of the above bacterial solution into 200 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.

3. Add 0.8 mM formaldehyde into each group when OD600 increased to 0.6 and the culture condition is the same as before.

4. Then, sampling culture in 96-well plate reader every 3 hours to measure the fluorescence intensity (ECFP) and corresponding OD600, then calculate the fluorescence / OD value of each group.

Here is the result:

caption

Fig 3. Fluorescence intensity (GFP) /OD expressed by weak promoter, J23100 and blank. Data are collected and analyzed according to iGEM standard data analysis form after 6 hours of induction.

Note: weak promoter is the promoter on the registry formaldehyde promoter (BBa_K1334002)

caption

Fig 4. The curve of fluorescence intensity (eCFP) /OD by pHCHO (BBa_K1334002) and formaldehyde_derivative-3 promoter.

In the former figure, we can see that the strong promoter BBa_J23100 group has a higher relative fluorescence intensity than the weak promoter present in the registry formaldehyde promoter, and in the latter figure, we can compare formaldehyde_derivative-3 promoter with the other two derivative promoters. We can conclude from the latter figure that formaldehyde_derivative-3 promoter is more sensitive to formaldehyde than the registry one. What’s more, formaldehyde_derivative-3 promoter is the most sensitive promoter among them. The detail about formaldehyde_derivative-1 promoter and formaldehyde_derivative-2 promoter can be seen in BBa_K3332042 and BBa_K3332043.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 422
    Illegal NheI site found at 445
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Reference

[1]Yurimoto, H., Hirai, R., Matsuno, N., Yasueda, H., Kato, N. and Sakai, Y. (2005), HxlR, a member of the DUF24 protein family, is a DNA‐binding protein that acts as a positive regulator of the formaldehyde‐inducible hxlAB operon in Bacillus subtilis. Molecular Microbiology, 57: 511-519. doi:10.1111/j.1365-2958.2005.04702.x